The beer, wine and baking yeast Saccharomyces cerevisiae has already been used for centuries for the production of bread, wine and beer owing to its characteristic of fermenting sugar to ethanol and carbon dioxide. In biotechnology, S. cerevisiae is used particularly in ethanol production for industrial purposes, in addition to the production of heterologous proteins. Ethanol is used in numerous branches of industry as an initial substrate for syntheses. Ethanol is gaining increasing importance as an alternative fuel, due to the increasingly scarce presence of oil, the rising oil prices and continuously increasing need for petrol worldwide. Moreover, S. cerevisiae is also used for the production of other biofuels or valuable biochemical compounds like isobutanol, succinic acid, farnesen/farnesan or artemisinin.
In order to make possible a favourably-priced and efficient biofuel production, the use of biomass containing lignocellulose, such as for example straw, waste from the timber industry and agriculture and the organic component of everyday household waste, presents itself as an initial substrate. Firstly, said biomass is very convenient and secondly is present in large quantities. The three major components of lignocellulose are lignin, cellulose and hemicellulose. Hemicellulose, which is the second most frequently occurring polymer after cellulose, is a highly branched heteropolymer. It consists of pentoses (L-arabinose, D-xylose), uronic acids (4-O-methyl-D-glucuronic acid, D-galacturonic acid) and hexoses (D-mannose, D-galactose, L-rhamnose, D-glucose). Although hemicellulose can be hydrolyzed more easily than cellulose, it contains the pentoses L-arabinose and D-xylose, which can normally not be converted by the yeast S. cerevisae. 
In order to be able to use pentoses for fermentations, these must firstly enter the cell through the plasma membrane. Although S. cerevisiae is not able to metabolize D-xylose or L-arabinose, it can take up D-xylose or L-arabinose into the cell. However, S. cerevisiae does not have a specific transporter. The transport takes place by means of the hexose transporters. The affinity of the transporters to D-xylose is, however, distinctly lower than to D-glucose (Kotter and Ciriacy, 1993). In yeasts which are able to metabolize D-xylose, such as for example P. stipitis, C. shehatae or P. tannophilus (Du Preez et al., 1986), there are both unspecific low-affinity transporters, which transport D-glucose, and also specific high-affinity proton symporters only for D-xylose (Hahn-Hagerdal et al., 2001).
In earlier experiments, some yeasts were found, such as for example Candida tropicalis, Pachysolen tannophilus, Pichia stipitis, Candida shehatae, which by nature ferment D-xylose or L-arabinose or can at least assimilate it. However, these yeasts lack entirely the capability of fermenting L-arabinose and D-xylose to ethanol, or they only have a very low ethanol yield (Dien et al., 1996). Moreover, very little is yet known about the uptake of D-xylose and L-arabinose. In the yeast C. shehatae one assumes a proton symport (Lucas and Uden, 1986). In S. cerevisiae, it is known from the galactose permease Gal2 that it can transport D-xylose but also transports L-arabinose, which is very similar in structure to D-galactose. (Kou et al., 1970). Most hexose transporters can mediate uptake of D-xylose.
Alcoholic fermentation of pentoses in biotechnologically modified yeast strains of S. cerevisiae, wherein inter alia various genes of the yeast strain Pichia stipitis were used for the genetic modification of S. cerevisiae, was described in recent years particularly in connection with the fermentation of xylose. The engineering concentrated here particularly on the introduction of the genes for the initial xylose assimilation from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, i.e. into a yeast which is traditionally used in the ethanol production from hexose (Jin et al. 2004).
Jeppson et al. (2006) describe xylose fermentation by S. cerevisiae by means of the introduction of a xylose metabolic pathway which is either similar to that in the yeasts Pichia stipitis and Candida shehatae, which naturally use xylose, or is similar to the bacterial metabolic pathway.
Katahira et al. (2006) describe sulphuric acid hydrolysates of lignocellulose biomass such as wood chips, as an important material for the production of fuel bioethanol. In this study, a recombinant yeast strain was constructed, which is able to ferment xylose and cellooligosaccharides. For this, various genes were integrated into this yeast strain and namely for the inter-cellular expression of xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae and for the presentation of beta-glucosidase from Aspergillus acleatus on the cell surface. In the fermentation of sulphuric acid hydrolysates of wood chips, xylose and cellooligosaccharides were fully fermented by the recombinant strain after 36 hours.
Pitkanen et al. (2005) describe the obtaining and characterizing of xylose chemostat isolates of a S. cerevisiae strain, which over-expresses genes of Pichia stipitis coding for xylose reductase and xylitol dehydrogenase and the gene which codes endogenous xylulokinase. The isolates were obtained from aerobic chemostat cultures on xylose as the single or major carbon source. Under aerobic conditions on minimal medium with 30 g/l xylose, the growth rate of the chemostat isolates was 3 times higher than that of the original strain (0.15 h−1 compared with 0.05 h−1). The xylose uptake rate was increased almost two-fold. The activities of the key enzymes of the pentose phosphate metabolic pathway (transketolase, transaldolase) were increased two-fold, whilst the concentrations of their substrates (pentose-5-phosphates, sedoheptulose-7-phosphate) were lowered accordingly.
Brat et al. (2009) screened nucleic acid databases for sequences encoding putative xylose isomerases and finally were able to clone and successfully express a highly active new kind of xylose isomerase from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize D-xylose and to use it as the sole carbon and energy source.
Demeke et al. (2013) developed an expression cassette containing 13 genes including C. phytofermentans xylA, encoding D-xylose isomerase, and enzymes of the pentose phosphate pathway and inserted the cassette in two copies in the genome of the industrial S. cerevisiae strain Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established highly efficient D-xylose fermentation.
Becker and Boles (2003) describe the engineering and the selection of a laboratory strain of S. cerevisiae which is able to use L-arabinose for growth and for fermenting it to ethanol. This was possible due to the over-expression of a bacterial L-arabinose metabolic pathway, consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous over-expression of yeast galactose permease transporting L-arabinose in the yeast strain.
Molecular analysis of the selected strain showed that the predetermining precondition for a use of L-arabinose is a lower activity of L-ribulokinase. However, inter alia, a very slow growth is reported from this yeast strain.
Wiedemann and Boles (2008) show that expressing of the codon-optimized genes of L-arabinose isomerase from Bacillus licheniformis and L-ribulokinase and L-ribulose-5-P 4-epimerase from Escherichia coli strongly improved L-arabinose conversion rates.
Farwick et al. (2014) developed a new system for screening and engineering of pentose transporters which are no longer inhibited by glucose. This system was based on a D-xylose-fermenting yeast strain having deletions of all hexose-transporters and all hexo-/glucokinases (hxt0 hxk0 strain). D-glucose can no longer be used as a carbon source but interferes with D-xylose utilization at transport level. As a result, mutant transporters that allow D-xylose uptake in the presence of increasing concentrations of D-glucose could easily be selected.
Using this system in evolutionary engineering and mutagenesis approaches the authors were able to generate specific D-xylose transporters from S. cerevisiae hexose transporters. Some of these mutant transporters had an exchange at a position corresponding to N376 of the galactose transporter Gal2. However, although they proved resistant against glucose most of them had a reduced uptake rate for xylose.
WO 2008/080505 A1 discloses an arabinose transporter from Pichia stipitis, which enables yeast cells to take up L-arabinose. EP 11 001 841.3 discloses a specific arabinose transporter of the plant Arabidopsis thaliana for the construction of pentose-fermenting yeasts. WO 2012/049170 A2 and WO 2012/049173 A1 disclose pentose and glucose fermenting yeast cells which contain and express among other nucleic acids, a polypeptide with arabinose permease activity comprising a mutation in position T219 to asparagine or N376 to serine of Gal2 which renders the transporter resistant against the inhibitory effect of glucose.
There still exists a need in the art for specific pentose transporters, in particular specific D-xylose transporters, which have a higher affinity and/or higher activity for pentoses, in particular combined with glucose resistance, which allow to specifically take up D-xylose and/or L-arabinose into cells, such as yeast cells, with high uptake rates even at low pentose concentrations, and therefore to promote the utilization and fermentation of pentoses, in particular D-xylose and/or L-arabinose, and in particular in the simultaneous presence of glucose.
It is thus an object of the present invention to provide improved and/or more specific transporters, which transport pentose(s), such as D-xylose and/or L-arabinose with higher activities and/or higher affinities.